Molecular response of chorioretinal endothelial cells to complement injury: implications for macular degeneration

Abstract

Age-related macular degeneration (AMD) is a common, blinding disease of the elderly in which macular photoreceptor cells, retinal pigment epithelium and choriocapillaris endothelial cells ultimately degenerate. Recent studies have found that degeneration of the choriocapillaris occurs early in this disease and that endothelial cell drop-out is concomitant with increased deposition of the complement membrane attack complex (MAC) at the choroidal endothelium. However, the impact of MAC injury to choroidal endothelial cells is poorly understood. To model this event in vitro, and to study the downstream consequences of MAC injury, endothelial cells were exposed to complement from human serum, compared to heat-inactivated serum, which lacks complement components. Cells exposed to complement components in human serum showed increased labelling with antibodies directed against the MAC, time- and dose-dependent cell death, as assessed by lactate dehydrogenase assay and increased permeability. RNA-Seq analysis following complement injury revealed increased expression of genes associated with angiogenesis including matrix metalloproteinase (MMP)-3 and -9, and VEGF-A. The MAC-induced increase in MMP9 RNA expression was validated using C5-depleted serum compared to C5-reconstituted serum. Increased levels of MMP9 were also established, using western blot and zymography. These data suggest that, in addition to cell lysis, complement attack on choroidal endothelial cells promotes an angiogenic phenotype in surviving cells.

Keywords: age-related macular degeneration; complement system; endothelial cells; matrix metalloproteinase.

Figure 1

High resolution detection of complement complexes in the human choriocapillaris. (A) Confocal microscopy of MAC (green) and UEA-I (red). Note the punctate MAC reactivity along the vitread surface of the choriocapillaris. Higher magnification (B) shows regional colocalization of MAC with the EC cell surface (arrows). (C) ImmunoEM localization of the membrane attack complex in human choroid. Section incubated with anti-MAC antibody shows labeling in the extracellular matrix of Bruch’s membrane (BrM); in addition, labeling is observed on a choroidal EC (arrow). (D) Section from the same eye incubated with secondary antibody alone. Scale bars = 10μm (A), 2.5μm (B), 500nm (D).

Figure 2

Immunocytochemistry of activated membrane attack complex on primate ECs. Labeling with an anti-MAC antibody (green) was remarkable on cells exposed to 50% normal serum (A), but labeling was not observed in the presence of heat-inactivated serum (B). Cells in C5-depleted serum failed to activate complement (C), unless the serum was reconstituted with C5 (D). Exposure and level adjustments were identical between A& B and C & D, respectively. Blue counterstain, DAPI. Scalebar = 100μm.

Figure 3

Cytolysis of ECs with complement-depleted and complement-containing serum. (A) Relative cell death of normal human serum-treated (5% and 10%) and heat-inactivated serum (HIS, 5%) over the course of 24hr. Compared to HIS treated cells, a higher RCD appeared in both serum-treated groups at all time points (p<0.05). (B) Relative cell death after 4 hours following exposure to medium alone, medium with Triton X-100 (defined as 100% cell death), 20% C5-deficient serum, 20% C5-deficient serum reconstituted with C5, 20% heat inactivated serum (HIS), and 20% normal human serum (NHS). RCD was significantly higher in cells exposed to complement. Horizontal line shows level of cell death estimated in medium-alone exposed cells. Error bars indicate standard deviation. * p <0.01.

Figure 4

EC permeability after exposure to complement-intact serum compared to complement-inactivated serum. A dose- and time-dependent increase in transendothelial flux (mL/cm²) was observed in RF/6A cells following complement injury. An increased flux occurred in 5% to 100% normal serum-treated cells compared to 5% HIS and serum-free medium after 24 hours of treatment (p<0.05).

Figure 5

Western blot of MMP9 (A) and zymography (B) following exposure of RF/6A cells to complement. Blot (A) shows reactivity of anti-MMP9 antibody to conditioned media of cells treated with 20% normal serum (NHS); conditioned media of cells treated with 20% heat inactivated serum (HIS); and extracts of cells following exposure to NHS and HIS, respectively. After 4 hours, a series of immunoreactive bands was significantly elevated in the conditioned medium of treated cells compared to controls exposed to complement depleted serum. (B) Zymography analysis of MMP activity using conditioned media from cells exposed to 50% normal serum or 50% HIS, or from medium alone that was not conditioned by EC (“50% NHS only”). Three individual samples are depicted for each group. All samples showed a band of gelatinase activity at approximately 65. An 88 kDa gelatin-cleaving band was observed only in the normal serum-treated cells, compared to both HIS conditioned medium and serum containing non-conditioned media control groups, after 4 hours.

Figure 6. Deposition of the membrane attack complex in an eye with choroidal neovascularization

Section from a 70 year old female donor, showing labeling of the MAC (green) and the vascular marker UEA-I (red). Nuclei are counterstained with DAPI (blue). Note the presence of choriocapillaris ghost vessels (arrows). In some cases, MAC is observed around vessels within the CNVM, consistent with findings of MAC as an activator of angiogenesis-associated genes. Right panel, adjacent section with omission of the primary antibody and lectin. Scale bar = 100 μm.